1. Field of the Invention
The present invention relates to a method and a device for the analysis of reaction media comprising one or more cells, this method and this device making it possible to perform an automated high-throughput analysis.
2. Description of the Related Art
Currently, in biological research, and in particular in the pharmaceutical field, attempts have been made to analyze the phenotype of cell populations, and more particularly the proteome, in response to one or more stimulations exerted on these cell populations, so as to evaluate the impact of these stimulations on the cells to which they are applied. This analysis requires tools for carrying out reactions on living reaction media (cell populations) and the analysis of these reaction media under conditions such that there is no (or as little as possible) distortion of the reaction medium between these two steps, so as to limit the loss or transformation of the information.
There exists, in this research field, an increasing demand in terms of sample processing throughput. An increasing number of molecules are available to be tested, and increasingly varied cell systems (tissues, networks, cells) are available to be studied.
There exists therefore a need for tools for analyzing living reaction media, which make it possible to study these reaction media with as much objectivity as possible and with a processing rate that is as high as possible.
The present invention, which allies the culturing of cell populations on matrix supports and analysis by mass spectrometry, makes it possible to satisfy these expectations.
At the current time, phenotypic screening is carried out essentially using calorimetric methods, or fluorescent or radioactive molecules. These methods require the labeling of specific molecules, which means that the protein(s) investigated must be known beforehand. The present invention does not require any prior knowledge of the expected modifications of the cells subsequent to the stimuli.
Many methods today make it possible to analyze more or less directly the phenotype of one or more cells with a more or less rapid throughput.
The analyses directly using cells all have an operating principle in common, but differ with respect to the signal analyzed at output, which represents the phenotype:                optical signal: fluorescence, luminescence, colorimetry.        radioactive signal: labeled molecules.        electrical signal: electrophysiology.        
These various analytical methods are generally implemented in a plastic well format (96 or 384 wells per plate) which also requires a large amount of reagents (0.1 to 0.5 ml per well in a 96-well plate) and allows only a moderate throughput.
In addition, the signal is not directly representative of the phenotype, but requires a calibration that is often complex.
It is, moreover, difficult to directly analyze cell secretions, except by means of antibody uptake methods.
Analysis of the various phenotypes is done only on the basis of a single parameter, a molecule that is known, and for which the intention is to verify whether it is present or not: it is necessary, at the start, to know which molecule is being sought. For example, when the intention is to study the presence of a molecule in the cell, it is necessary, at the start, to have quite a precise idea of the various properties of this molecule, so as to couple it to a specific antibody or render the molecule fluorescent. It is also possible to look for post-translational modifications of proteins, but by targeting a specific modification.
Research relating to the limit of the performance and processing rate of these various methods has led to the development of a format that is more suited to very high-throughput handling. The first example of the result of this research is DNA chips: the messenger RNAs expressed by a cell population (after stimulation) are screened in the form of deposits on a matrix support, or chip, containing DNA fragments potentially complementary to this expression. This method makes it possible to assess the level of expression of several thousand genes on a chip.
However, the results are often not demonstrative enough to be able to do without a finer study after a first analysis. In fact, this method involves sample processing steps (collection and multiplication of the amount of RNA, reverse PCR) that move away from the cellular model under consideration. In addition, it only makes it possible to analyze the level of expression of RNA, which is not directly related to the amount of proteins produced, nor to the qualities thereof, due in particular to post-translational modifications (alternative splicing, modified quaternary 3D conformation, phosphorylation, assembly).
Other molecule chips attempt to overcome this distancing by trying to directly analyze the level of expression of various molecules within the cell culture. Thus, various molecules have been deposited in the form of a matrix on supports in the chip format: RNA, proteins, sugars, for example. Unfortunately, the techniques used are not really reliable, and are intended to analyze interactions between molecules rather that a cell phenotype.
Finally, even though the number of different molecules analyzed on a chip is large, a choice has already been made with regard to the molecules that are deposited onto the support. This approach implies prior knowledge of the phenotypes being sought, which restricts the analytical possibilities.
As regards cell chips, they have been described by Sabatini et al. These cell chips function in the following way: DNA is deposited in the form of a dispersion in gelatin, as a matrix, on a glass slide. After drying, the positions comprising DNA are treated with a lipid based transfection agent and the plate is then placed in a medium into which cells are dispensed.
On the glass slide, the gelatinized DNA is present in solid form and the transfection takes place in a semi-solid phase, by binding the molecules adjacent to the DNA deposits to lipids that promote penetration of the DNA into the cells adjacent to the DNA deposits. A matrix of transfected cells at the positions corresponding to the DNA deposits is obtained. This method has the drawback of not being very precise and of being nonreproducible. The attachment by the gelatin does not make it possible to control the detachment of the transfected DNA. Neither does it make it possible to improve the transfection efficiency. Using this method, the expression or the blocking of the expression of a sufficient amount of protein is difficult to obtain. Only one type of cell can be used for each glass slide. It is not possible, by this means, to study the interaction between cells or the interaction between cells and reagents other than DNA.
Compared with the analytical methods listed above, mass spectrometry constitutes a tool that is much more advantageous in terms of the relationship between the signal detected and the cell phenotype (the molecules expressed by the cell and their respective amounts). It in fact makes it possible to directly measure the amount of proteins present in a sample, without it being necessary to modify the integrity of the molecule (by labeling of the molecule, for example). The principle consists in desorbing from a solid support and ionizing molecules of the sample to be analyzed. Then, the mass/charge ratio of the particles thus created is recorded; it represents the signature of the desorbed molecule.
In the use currently made thereof in cell biology and in proteomics, many sample treatments are necessary (cell lysis, sample purification, introduction of a matrix for MALDI spectrometry, for example), because the specificity and the accuracy allowed for the moment by the instrumentation does not permit a direct analysis of the samples, the quality of which is not controlled to a high enough degree. These treatments introduce biases into the analysis, which is no longer really representative of the cell phenotype.
In addition, the implementation of these methods and the use of the existing equipment allows only a very low sample treatment throughput.
A new system (SELDI for “surface enhanced laser desorption ionization”) for using mass spectrometry on more or less complex samples that is a little closer to the cell model, has recently been developed. The asset of this system comes from its ability to perform some of the purifications necessary for obtaining a correct analysis, directly on the support which goes into the machine (selective and oriented adsorption at the surface of the support), which makes it possible to shorten and simplify the sample treatments.
This system is used in particular in the discovery of a disease marker: samples from a sick population are analyzed with respect to others from a normal population, and the differences are analyzed by means of a proprietary program; this makes it possible to identify markers for the disease, and therefore potentially advantageous targets. This method makes it possible to perform really an overall analysis of the samples: the molecules that will be the markers for the disease are not known beforehand.
However, sample treatments are still necessary (cell lysis, washing and purification) in order to obtain good results. For example, the cells are cultured away from the substrate, which implies a non-controlled sample transfer bias. Moreover, the analytical throughput is still low: the chips that can be used in this system can contain only 16 different plots to date.
Several documents concerning mass spectrometry relate to the analysis of living reaction media:                Document US-2002/0160420 describes the analysis by mass spectrometry of a sample of human serum that has undergone several purification steps.        Document US-2002/0076739 describes a method for analyzing proteins in mixtures. Labeled reagents specific for certain peptides are reacted with protein mixtures, and the molecules that have reacted are isolated and then analyzed by mass spectrometry.        Document DE 10038684 describes a method for identifying microorganisms using a MALDI-TOF-MS system, in which the spectrum of a sample of a microorganism to be identified is compared with a database of reference spectra.        Document U.S. Pat. No. 6,531,318 describes a method for analyzing biological tissues, this method comprising a step consisting of microdissection by means of a laser, this microdissection making it possible to select cell aggregates, followed by mass spectrometry analysis.        Document WO 00/48004 describes a device for analyzing cellular material. Cells are cultured, purified by methods other than chromatography, and then injected into a mass spectrometer.        
This method requires handling of the cell culture samples; in particular, the purification is a step in the method which eliminates certain constituents of the sample, without the selection of the components that are eliminated being completely controlled.                Document WO 02/103360 describes a method for analyzing proteins at the surface of a cell; this method comprises reacting the cell with a substance at its surface and analyzing it by mass spectrometry.        Document WO 01/65254 describes a method for identifying the chemical structure of a substance present in tissues or cells of various organisms, this method comprising the irradiation of a precise area of a section of living tissue or of a cell, so as to ionize the substance and determine its mass spectrum, and the analysis of this spectrum so as to identify its structure.        Document WO 02/101356 describes a method for analyzing mitochondrial proteins. The proteins constituting the mitochondrion are separated by two-dimensional gel electrophoresis and then analyzed by mass spectrometry.        Document WO 01/84143 describes a method for analyzing a large number of proteins in a small amount of time. Cells are subjected to a stimulation and lyzed, and the samples are divided up so as to obtain batches of a few hundred proteins, and these batches are then analyzed by mass spectrometry using a battery of spectrometers in parallel.        
The methods for analyzing living reaction media by mass spectrometry all comprise one or more purification and/or handling steps that result in a loss of information, and/or they imply the search for well-defined molecules, whereas one of the objectives of the invention was that of obtaining an analysis without any constraints regarding the data expressed by the living reaction medium.
In addition, these methods of the prior art, because of the purification and/or handling steps that they comprise, are not very suitable for high-throughput treatment.